As forests around the world have become depleted by logging for lumber, fuel, and land expansion, intensively managed tree plantations in the world's developed countries have become the major source for the world's sustainable supply of soft-woods. About eight species now comprise the great bulk of the plantation wood presently being grown worldwide. The predominant species in North America is usually one that is native to the region. In other areas of the world it is more typically an exotic that has proved particularly well adapted to the locale. The Monterey pine (Pinus radiata Don.) grown widely in Africa, Australia and New Zealand is an example of an exotic species which grows particularly well in a non-native locale.
Genetic selection of the plantation species has resulted in trees having heritable improvements in a number of regards in comparison with those found in natural stands. Rapid growth to harvestable size has been the principal improvement sought. This selection process has been so successful that in some areas rotations as low as 25 years are standard. Virtually all plantations are now restocked with seedlings grown from seed obtained from what in many cases is third generation seed orchards, In recent years large numbers of rooted cuttings from young trees originating from genetically select seed have also become an important source for restocking programs. This is one way of bulking up scarce and expensive full sib seed.
Rooted cuttings are an example of forestry where, on a small scale, the characteristics of selected parents are passed on intact to a succeeding generation. They have the disadvantage of being quite expensive in comparison with natural seedlings. For the past two decades research has been conducted on reproduction of conifers by tissue culture as a method of producing select clonal stock. This method is just now in its commercial infancy. The process most widely employed is embryogenesis. An embryo from a desirable seed is placed on a culture medium where multiple early stage genetically identical replicates are produced. Immature early stage embryos are placed on a series of media where they are further multiplied and cultured to a mature state where they are morphologically similar to zygotic embryos. These newly grown somatic embryos may then be placed on a germination medium for conversion into plants. Alternatively, they may be formed into manufactured seeds.
Some examples showing conifer embryogenesis procedures are found in U.S. Pat. Nos. 4,957,866, and 5,036,007 to Gupta et al., U.S. Pat. No. 5,034,326 to Pullman et al., U.S. Pat. No. 5,563,061 to Gupta, U.S. Pat. No. 5,413,930 and U.S. Pat. No. 5,506,136 to Becwar et al. and U.S. Pat. No. 5,187,092 to Uddin.
During the earlier tissue culture efforts the embryos produced had a very low success rate for conversion into rooted plants. This remains a problem today although the current success rate is much higher. However, the important Pine species have been particularly intractable. In the effort to increase successful conversion, much attention was given to culture conditions attempting to improve the morphology of the somatic embryos so that they physically resembled zygotic embryos as closely as possible. Various changes were made in the culture media nutrients and hormones to effect these improvements. Unfortunately, a high degree of morphological resemblance did not ensure good germination and conversion. More recently, investigators have studied the importance of storage products in somatic embryos as they relate to germination success and resulting plant vigor. Storage products are generally defined as lipids and proteins found within the embryo and in the surrounding megagametophyte of a natural seed. Some authorities in the field would also include carbohydrates as a component of storage products.
Storage products provide the initial energy needed upon germination. Additionally, the storage products may be associated with desiccation tolerance in embryos. One example of the importance placed on high levels of stored lipids in somatic embryos can be found in U.S. Pat. No. 5,464,769 to Attree et al.
In the discussion that follows, reference to journal articles are noted only by lead author and date. Reference should be made to the bibliography following the specification for full citations.
The biochemical changes that occur within developing somatic embryos are extremely complex and are still poorly understood. In addition to the lipid and proteinaceous materials, the carbohydrates now appear to have a critically important role. These have been studied for a number of species. Steadman et al. (1996) studied development of soluble sugars in a broad spectrum of angiosperm species. In particular, they looked at differences between "orthodox" seeds having high germination success and "recalcitrant" seeds which had poor germination. They found that orthodox seeds generally had significantly higher ratios of the oligosaccharides raffinose and stachyose to sucrose at maturity. Frias et al. (1996) studied three legume species. They noted that simple sugars decreased during seed development and raffinose, stachyose and verbascose appeared later as the seed matured. They did not specifically study embryos and found significant differences between species. Black et al. (1996), reported their work with development of wheat embryos. An early starch accumulation declined to a very low value at maturation Sucrose and raffinose continued to increase during maturation, the major increase in raffinose approximating the time of the fall in starch content. The development of desiccation tolerance was associated with increasing raffinose to sucrose ratios. Bernal-Lugo et al. (1992), note that depletion of raffinose in aged corn is related to a decline in seed vigor.
Workers in the field of tissue culture learned early on that it was an inexact and unpredictable science. What worked for one genus failed for another. Often what worked for one species failed for a closely related species within the same genus. The correspondence gap has been particularly wide between the angiosperms and gymnosperms. To the present inventors' knowledge similar studies to those noted above on embryo development have not been carried out on the gymnosperm species within the botanical Order Coniferales. Studies of sugar content and metabolism have been carried out later in the process; i.e., on germinating seeds. Hattori et al. (1951) note the presence of sucrose, raffinose, and stachyose in mature seeds of Pinus thunbergii. As the tip of the young root appeared the raffinose and stachyose rapidly disappeared. Durzan et al (1968) examined the above three sugars and free amino acids in the embryos and female gameteophytes of jack pine (Pinus banksiana Lamb.). Geographic source of the seed introduced considerable variation in both the absolute levels of the three sugars as well as the respective ratios of the higher oligosaccharides to sucrose. Murphy et al. (1988) reported the levels of soluble sugars and hydrolytic enzymes as related to the release of dormancy and germination for sugar pine (Pinus lambertiana Dougl.). They noted that on germination, raffinose and stachyose dropped steadily to very low levels over about 15 days. Sucrose rose to a sharp peak at about 7 days then began a marked decline. Lin et al. (1994), in a study of 17 species including four Asiatic conifers, concluded that the ratio of oligosaccharide to disaccharide plays a role in desiccation tolerance and longevity of orthodox seeds. They note that the accumulation of the raffinose series of sugars is induced by slow drying during seed maturation but that the ratio between raffinose and stachyose is probably species dependent. Similarly, Leprince et al. (1993) state that oligosaccharides are important in cell wall protection of angiosperms during desiccation but conclude that they are only one of a suite of important and interrelated factors. Ching (1966) looked at the compositional changes in Douglas-fir during germination and concluded that the metabolic changes observed were similar to angiosperm seeds. Kao (1973) studied germination of Taiwan red pine (Pinus taiwanensis Hayata) and Chinese fir (Cunninghamia lanceolata (Lamb) Hook.) with the conclusion that fats were the main reserve materials. He noted that sucrose, raffinose and stachyose occurred in non-germinated seed of red pine while the oligosaccharides were replaced by fructose and glucose in germinated seeds. Raffinose, sucrose, fructose and glucose were found in both non-germinated and germinated seed of Chinese fir.
In addition to the di- and oligosaccharides formed in developing embryos, a group of extremely hydrophilic, heat-soluble proteins with no enzyme activity called Late Embryogenesis Abundant (LEA) proteins accumulates in plant embryos (Dure et al. 1989). Within this group of proteins is a family generally termed "dehydrins" (Close et al. 1993). Genes for dehydrins are expressed (1) naturally during seed development (Close et al. 1993); (2) in response to cold and water stress (Hurkman et al. 1996; Wisniewski et al. 1996); and (3) in response to the phytohormone abscisic acid (ABA). Although both ABA and water stress play important roles during seed development, it is not clear what signal induces the synthesis of dehydrin during seed development in situ (Han et al 1996: Wood et al. 1997).
Neither is the function of the LEA family of proteins entirely clear. But, because of its pattern of expression, it is thought to be involved in stress tolerance. Nevertheless, attempts to define a precise function in tolerance to desiccation or cold have proven fruitless and the search for function still goes on. In angiosperm zygotic embryos, dehydrin proteins accumulate during late embryogenesis--after the major period of reserve deposition is completed (Han et al. 1997). They can also be prematurely induced under a variety of conditions upon excision of young embryos from the mother plant (Galau et al. 1991).
Germination of embryos is an outcome of cell expansion and cell division. The first visible sign of germination in isolated embryos is axis elongation (radicle+hypocotyl+epicotyl). After an embryo is placed in an environment with a water potential high enough to support germination (greater than about -2.0 MPa) it hydrates to a certain water content. No further visible changes occur until germination itself The period between placing the embryo on water and visible germination is referred to as "lag time". In a mature seed of a given species, the length of the lag time can be closely predicted. It depends on water potential, endogenous ABA, and temperature. It may be that certain biochemical events must occur before cell expansion leading to germination can occur and that the rate of these biochemical events depends on water content and temperature. Respiratory rate during this time is a function of water content, temperature, and time. It has been noted that respiratory rate increases with imbibition time. However, respiration appears to be indicative of biochemical reaction in general rather than causative of germination. From the fact that respiration and utilization of stored reserve products occurs during the lag phase, it follows that the longer the lag phase at a given temperature and water content, the less stored reserves remain for early seedling growth.
If embryos or seeds are excised before a critical point in their development they may not germinate at all. If they do, they do so slowly and often exhibit abnormalities (e.g., see Blackman et al. 1992). An unusually long lag time contributes to the poor vigor in these cases. It seems reasonable that the young embryos are using this lag time to complete some unfulfilled biochemical process that is essential for them to become germinable--a process that would have otherwise occurred had they been left to complete their normal development on the mother plant.
A candidate for this putative process is protein synthesis. Gene expression studies show that when immature angiosperm embryos of a number of species are excised and placed on nutrient medium, two groups of proteins are synthesized (Jacobsen et al. 1994). One group, consisting of enzymes for reserve breakdown, is characteristic of germination. The other group is the LEA proteins. Concomitant with the synthesis of these proteins, storage proteins are catabolized.
The co-expression of two developmental programs that are normally temporally distinct during zygotic embryogenesis may have profound implications for the vigor of the germinant. If certain developmental events must be completed before germination can occur, then it is likely that the prolonged lag phase in immature embryos reflects the time necessary to complete these events. However, if germination (at least in the sense of reserve breakdown) starts before these events are completed, the embryo, when it is finally ready to germinate, is left with less "fuel" for subsequent growth since it was used during the prolonged lag phase. Left to develop on the plant, the continuous withdrawal of water from the system ensures that the required developmental events will be completed in a timely manner without the premature onset of germination.
It has been frequently shown that incubating young embryos at a water potential that does not permit germination, but is still high enough to permit biochemical activity, decreases the lag time so that it approaches that of mature embryos (e.g., see Blackman et al. 1992). During this time specific proteins are synthesized including heat soluble proteins and dehydrin (Han et al. 1997). Incubation at high R.H., causing a concomitant slow drying, has also been shown to enhance germinability in gymnosperm somatic embryos, (e.g., Roberts, U.S. Pat. No. 5,183,757, Roberts et al. 1990). One might also predict that the slightly lowered water potential prevents the hydrolysis of reserves that would occur at higher water potentials so that the embryos can complete the developmental steps necessary for germination without compromising their reserve status.
The heat soluble proteins, including dehydrin, that are abundant during maturation and quiescence are rapidly broken down during germination whether the embryos are naturally matured or prematurely dried. The tight link between quiescence or dormancy and the presence of dehydrin has been noted both in seeds and non-seed dormant tissue such as overwintering buds (Wisniewski et al. 1996).
In zygotic embryogenesis, "maturity" is easy to identify because the seed dries and dehisces from the mother plant Shortly after the onset of drying the zygotic embryo attains maximum germinability. Essentially, the pre-programmed development and environmental responses of the embryo and mother plant dictate maturity. We are left simply to harvest the mature seed and treat it optimally after harvest. However, this is not the case with somatic embryos where scientists must dictate the timing and protocol of every shift in hormones, media composition, water potential, photoperiod, and temperature. In somatic embryos, the period of quiescence which so clearly demarcates maturation from germination in zygotic embryogenesis is completely lacking. Morphological maturity based on appearance has heretofore been used as a criterion but this crude tool has proved to be highly undependable. Other tools or markers which would serve in its stead, to signal the achievement of maximum maturity and readiness for germination, have heretofore been lacking.
The requirements of an embryo during maturation are completely different and virtually opposite to the requirements of an embryo during germination. Morphology is the outcome or result of changes that have taken place at the biochemical level. However, it does not reveal all of them, particularly at this critical juncture between biochemical maturity and readiness to germinate. More precise biochemical tools to signal these changes would be extremely helpful to the scientists working with somatic embryogenesis. It would allow them to precisely identify needed protocol changes and the timing of their imposition.
None of the investigators working on conifer tissue culture appear to have looked at the development over time of the more complex sugars in maturing somatic embryos nor has the importance of this been recognized. Neither do they have seemed to study in any detail the development of the dehydrin protein group and its importance.